RING E3 ubiquitin ligases are specific binding proteins that mediate the reaction of a ubiquitinated ubiquitin conjugating enzyme (E2-Ub) with a target protein to transfer ubiquitin from the active-site Cys of the E2 to a Lys side chain of the target. Because target proteins are frequently modified by several equivalents of ubiquitin, RING E3 ligases bind E2-Ub multiple times. Second and subsequent transfers can occur on different Lys residues, producing a multiply monoubiquitinated target, or to a Lys residue of a previously transferred ubiquitin, producing a polyubiquitinated target (Passmore and Barford, 2004). By gel electrophoresis, multiple monoubiquitination and polyubiquitination both appear as ladders in which the target protein is modified by ubiquity! units of ~8 kDa, yet the products of the reaction are distinct. Novel quantitative mass spectrometric methods have recently been developed to solve the analytical protein chemistry part of this problem (Kirkpatrick et al., 2005a;Kirkpatrick et al., 2005b), though there have been few integrated efforts to define precisely what RING E3 ubiquitin ligases do using a combination of mass spectrometry, enzymology, cell biology and genetics. The complexity of target protein ubiquitination is due to multiple factors. First, target proteins have multiple Lys residues that are solvent-exposed and potentially additional Lys residues that become accessible after denaturation or initial cycles of ubiquitination. All such Lys residues are potential primary targets of ubiquitination. Second, ubiquitin has 7 Lys residues, all of which are ubiquitinated in yeast extracts, with the principle sites of polyubiquitin linkage at Lys48 and Lys63 (Peng et al., 2003;Kirkpatrick et al., 2005a). Third, the E2 ubiquitin conjugating enzymes have different specificities and potentially form different sets of target protein and polyubiquitin linkages in combination with different E3 ubiquitin ligases, which also have distinct specificities. Fourth, many RING E3 ligases, which comprise just one class of E3 ligases, have been proposed to have biologically important autoubiquitination reactions in addition to, or to the exclusion of, modification of external substrates. For example, evidence has been presented that Chfr, a human RING E3 ubiquitin ligase that is inactivated in 20-50% of human tumors (Scolnick and Halazonetis, 2000;Mizuno et al., 2002;Shibata et al., 2002;Corn et al., 2003;Mariatos et al., 2003;Toyota et al., 2003), functions with Ubc13/Mms2 to modify itself with Lys63-linked polyubiquitin to signal cellular stress (Bothos et al., 2003;Matsusaka and Pines, 2004). Evidence has also been presented that Chfr catalyzes Ubc4 and Ubc5-dependent ubiquitination of Polo-like kinase 1 (Plk1) leading to proteasomal degradation of Plk1 to block cell cycle transition (Kang et al., 2002). In the case of Chfr, does function depend on autoubiquitination or substrate-ubiquitination or both? What are the sites and linkages? Which E2s are really involved? What is the collection of substrates that are modified? To determine the biochemical and cellular mechanisms of function of Chfr-related molecules, it became necessary to develop genetic systems with which to dissect RING E3 ligase functions (Bieganowski et al., 2004), to reconstitute purified systems to characterize the determinants and products of the reactions, and to refine mass spectrometric methods to identify and to quantify sites and linkages. The applicant and coapplicant have developed these systems, combining their expertise in genetic, biochemical and mass spectrometric analysis in work made possible by limited discretionary funding. Yeast Chfr homologs, Chf1 and Chf2, have been cloned, characterized genetically, purified, and used to reconstitute cell-free ubiquitination reactions, which have been analyzed by mass spectrometry. The reaction components have also been validated genetically and the "interactomes" of Chf1 and Chf2 have been determined. Specific Aims: 1) We will use quantitative mass spectrometry and enzymology to define the sites, linkages and kinetics of Chfl and Chf2 ubiquitination reactions with genetically validated ubiquitin conjugating enzymes and the proteins we have identified as Chf interactors. 2) We will determine the sites, linkages, biological consequences, and E2-dependence of Chf1 and Chf2 ubiquitination in vivo. This proposal has two long-term public health objectives. First, determining the mechanisms of action of yeast Chf1 and Chf2 is critical to understand the function of Chfr, which is frequently inactivated in human tumors of epithelial origin. Second, innovations in analysis of RING E3 ubiquitin ligases are necessary to understand the specificity of function of RING E3 ubiquitin ligases, which have key functions in the health of every organ system.